System-wide approaches to uncover Th2 cell lineage commitment

T helper (Th) cells are vital regulators of the adaptive immune system. When activated by presentation of cognate antigen, Th cells demonstrate capacity to differentiate into functionally distinct effector cell subsets. The Th2 subset is required for protection against extracellular parasites, such as helminths, but is also closely linked to pathogenesis of asthma and allergies. The intracellular molecular signal transduction pathways regulating T helper cell subset differentiation are still incompletely known. Moreover, great majority of studies regarding Th2 differentiation have been conducted with mice models, while studies with human cells have been fewer in comparison. The goal of this thesis was to characterize molecular mechanisms promoting the development of Th2 phenotype, focusing specifically on human umbilical cord blood T cells as an experimental model. These primary cells, activated and differentiated to Th2 cells in vitro, were investigated by complementary system-wide approaches, targeting levels of mRNA, proteins, and lipid molecules. Specifically, the results indicated IL4-regulated recruitment of nuclear protein, and described novel components of the Th2-promoting STAT6 enhanceosome complex. Furthermore, the development of the activated effector cell phenotype was found to correlate with remodeling of the cellular lipidome. These findings will hopefully advance the understanding of human Th2 cell lineage commitment and development of Th2-associated disease states.Siirretty Doriast

A practical guide to single-cell RNA- sequencing for biomedical research and clinical applications

RNA sequencing (RNA-seq) is a genomic approach for the detection and quantitative analysis of messenger RNA molecules in a biological sample and is useful for studying cellular responses. RNA-seq has fueled much discovery and innovation in medicine over recent years. For practical reasons, the technique is usually conducted on samples comprising thousands to millions of cells. However, this has hindered direct assessment of the fundamental unit of biology—the cell. Since the first single-cell RNA-sequencing (scRNA-seq) study was published in 2009, many more have been conducted, mostly by specialist laboratories with unique skills in wet-lab single-cell genomics, bioinformatics, and computation. However, with the increasing commercial availability of scRNA-seq platforms, and the rapid ongoing maturation of bioinformatics approaches, a point has been reached where any biomedical researcher or clinician can use scRNA-seq to make exciting discoveries. In this review, we present a practical guide to help researchers design their first scRNA-seq studies, including introductory information on experimental hardware, protocol choice, quality control, data analysis and biological interpretation.  </div

Single-cell characterization of dog allergen–specific T cells reveals TH2 heterogeneity in allergic individuals

BackgroundAllergen-specific type 2 CD4+ TH2 cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the TH2 response has only recently been appreciated.ObjectiveWe sought to characterize at the single-cell level the ex vivo phenotype, transcriptomic profile, and T-cell receptor (TCR) repertoire of circulating CD4+ T cells specific to the major dog allergens Can f 1, Can f 4, and Can f 5 in subjects with and without dog allergy.MethodsDog allergen–specific memory CD4+ T cells were detected ex vivo by flow cytometry using a CD154-based enrichment assay and single-cell sorted for targeted gene expression analysis and TCR sequencing.ResultsDog allergen–specific T-cell responses in allergic subjects were dominantly of TH2 type. TH2 cells could be phenotypically further divided into 3 subsets, which consisted of TH2-like (CCR6−CXCR3−CRTH2−), TH2 (CCR6−CXCR3−CRTH2+CD161−), and TH2A (CCR6−CXCR3−CRTH2+CD161+CD27−) cells. All these subsets were nonexistent within the allergen-specific T-cell repertoire of healthy subjects. Single-cell transcriptomic profiling confirmed the TH2-biased signature in allergen-specific T cells from allergic subjects and revealed a TH1/TH17 signature in nonallergic subjects. TCR repertoire analyses showed that dog allergen–specific T cells were diverse and allergic subjects demonstrated less clonality compared to nonallergic donors. Finally, TCR and transcriptomic analyses revealed a close relationship between TH2-like, TH2, and TH2A cells, with the last ones representing the most terminally differentiated and highly polarized subtype.ConclusionsOur study demonstrates heterogeneity within allergen-specific TH2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches.</p

Single-cell characterization of leukemic and non-leukemic immune repertoires in CD8(+) T-cell large granular lymphocytic leukemia

T cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder of mature, clonally expanded T cells, where somatic-activating STAT3 mutations are common. Although T-LGLL has been described as a chronic T cell response to an antigen, the function of the non-leukemic immune system in this response is largely uncharacterized. Here, by utilizing single-cell RNA and T cell receptor profiling (scRNA+TCR alpha beta-seq), we show that irrespective of STAT3 mutation status, T-LGLL clonotypes are more cytotoxic and exhausted than healthy reactive clonotypes. In addition, T-LGLL clonotypes show more active cell communication than reactive clones with non-leukemic immune cells via costimulatory cell-cell interactions, monocyte-secreted proinflammatory cytokines, and T-LGLL-clone-secreted IFN gamma. Besides the leukemic repertoire, the non-leukemic T cell repertoire in T-LGLL is also more mature, cytotoxic, and clonally restricted than in other cancers and autoimmune disorders. Finally, 72% of the leukemic T-LGLL clonotypes share T cell receptor similarities with their non-leukemic repertoire, linking the leukemic and non-leukemic repertoires together via possible common target antigens. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clonotype. T cell large granular lymphocytic leukemia (T-LGLL) is a lymphoproliferative disorder involving clonally expanded T cell clones and is not fully understood. Here the authors show that the rest of the immune repertoire is interconnected with the T-LGLL clonotype(s) and is more mature, cytotoxic and clonally restricted than in other cancers and autoimmune disorders.Peer reviewe

Evolution and modulation of antigen-specific T cell responses in melanoma patients

Analyzing antigen-specific T cell responses at scale has been challenging. Here, we analyze three types of T cell receptor (TCR) repertoire data (antigen-specific TCRs, TCR-repertoire, and single-cell RNA + TCRαβ-sequencing data) from 515 patients with primary or metastatic melanoma and compare it to 783 healthy controls. Although melanoma-associated antigen (MAA) -specific TCRs are restricted to individuals, they share sequence similarities that allow us to build classifiers for predicting anti-MAA T cells. The frequency of anti-MAA T cells distinguishes melanoma patients from healthy and predicts metastatic recurrence from primary melanoma. Anti-MAA T cells have stem-like properties and frequent interactions with regulatory T cells and tumor cells via Galectin9-TIM3 and PVR-TIGIT -axes, respectively. In the responding patients, the number of expanded anti-MAA clones are higher after the anti-PD1(+anti-CTLA4) therapy and the exhaustion phenotype is rescued. Our systems immunology approach paves the way for understanding antigen-specific responses in human disorders.</p

Evolution and modulation of antigen-specific T cell responses in melanoma patients

Analyzing antigen-specific T cell responses at scale has been challenging. Here, we analyze three types of T cell receptor (TCR) repertoire data (antigen-specific TCRs, TCR-repertoire, and single-cell RNA + TCR alpha beta-sequencing data) from 515 patients with primary or metastatic melanoma and compare it to 783 healthy controls. Although melanoma-associated antigen (MAA) -specific TCRs are restricted to individuals, they share sequence similarities that allow us to build classifiers for predicting anti-MAA T cells. The frequency of anti-MAA T cells distinguishes melanoma patients from healthy and predicts metastatic recurrence from primary melanoma. Anti-MAA T cells have stem-like properties and frequent interactions with regulatory T cells and tumor cells via Galectin9-TIM3 and PVR-TIGIT -axes, respectively. In the responding patients, the number of expanded anti-MAA clones are higher after the anti-PD1(+anti-CTLA4) therapy and the exhaustion phenotype is rescued. Our systems immunology approach paves the way for understanding antigen-specific responses in human disorders.Peer reviewe

Single-Cell Epigenomics and Functional Fine-Mapping of Atherosclerosis GWAS Loci

Rationale: Genome-wide association studies have identified hundreds of loci associated with coronary artery disease (CAD). Many of these loci are enriched in cisregulatory elements but not linked to cardiometabolic risk factors nor to candidate causal genes, complicating their functional interpretation. Objective: Single-nucleus chromatin accessibility profiling of the human atherosclerotic lesions was used to investigate cell type-specific patterns of cisregulatory elements, to understand transcription factors establishing cell identity, and to interpret CAD-relevant, noncoding genetic variation. Methods and Results: We used single-nucleus ATAC-seq (assay for transposase-accessible chromatin with sequencing) to generate DNA accessibility maps in >7000 cells derived from human atherosclerotic lesions. We identified 5 major lesional cell types including endothelial cells, smooth muscle cells, monocyte/macrophages, natural killer/T cells, and B cells and further investigated subtype characteristics of macrophages and smooth muscle cells transitioning into fibromyocytes. We demonstrated that CAD-associated genetic variants are particularly enriched in endothelial and smooth muscle cell-specific open chromatin. Using single-cell coaccessibility and cis-expression quantitative trait loci information, we prioritized putative target genes and candidate regulatory elements for approximate to 30% of all known CAD loci. Finally, we performed genome-wide experimental fine-mapping of the CAD variants identified in genome-wide association studies using epigenetic quantitative trait loci analysis in primary human aortic endothelial cells and self-transcribing active regulatory region sequencing (STARR-Seq) massively parallel reporter assay in smooth muscle cells. This analysis identified potential causal single-nucleotide polymorphisms (SNPs) and the associated target gene for over 30 CAD loci. We present several examples where the chromatin accessibility and gene expression could be assigned to one cell type predicting the cell type of action for CAD loci. Conclusions: These findings highlight the potential of applying single-nucleus ATAC-seq to human tissues in revealing relative contributions of distinct cell types to diseases and in identifying genes likely to be influenced by noncoding genome-wide association study variants.</p